Project Summary and Abstract: The goal of our proposed studies is to elucidate the expression profiles and molecular functions of short non-coding RNAs (ncRNAs) in the pathogenesis of asthma. Asthma is a chronic inflammatory disease characterized by inflammation, mucus production, airway re-modeling, and hyper- responsiveness. In asthma, allergic inflammatory mediators, such as cytokines, act on resident airway cells [airway smooth muscle (ASM) cells, epithelial (AE) cells and fibroblasts (LF)] causing their structural and functional changes. However, knowledge gaps remain in our understanding of the mechanisms by which inflammatory mediators modulate cellular phenotypes. Although transcriptional regulation of gene expression in resident airway cells has been extensively studied, regulatory mechanisms at post-transcriptional steps remain elusive. In this context, short ncRNAs have evolved as one of the key post-transcriptional regulators of gene expression. Previous transcriptome profiling for short ncRNA analyses relied mainly on standard RNA-seq methods which fail to detect many RNA species. Cyclic phosphate-containing RNAs (cP-RNAs), that harbor a cyclic phosphate (cP) at their 3?-end, are one such RNA species not captured by RNA-seq, because cP prevents 3?-adapter ligation. Their absence in RNA-seq data makes cP-RNAs an invisible, hidden component of transcriptomes. Importantly, cP-RNAs are expressed as functional molecules. For example, angiogenin- generated 5?-tRNA halves, containing a cP and thus belonging to cP-RNAs, have functional significance in stress response, translational regulation, and cell proliferation, and are associated with neurodegenerative diseases and cancers. We propose that 5?-tRNA halves and other cP-RNAs play important roles in asthma pathobiology. In preliminary studies, we found that mouse lung expresses specific 5?-tRNA halves and cP-RNAs whose levels are upregulated during allergic inflammation caused by inhaled challenge of house dust mite (HDM). Furthermore, in human ASM cells, 5?-tRNA halves function to regulate cellular focal adhesion, migration, and proliferation. These results allowed us to hypothesize that inflammatory mediators upregulate the levels of 5?- tRNA halves/cP-RNAs, which contributes to airway cellular phonotypic changes in the molecular pathogenesis of asthma. By using our developed cP-RNA-seq, we propose to fully elucidate the regulation of the expression of 5?-tRNA halves/cP-RNAs mediated by asthmatic conditions in human lung and plasma samples and in human ASM cells, AE cells, and LFs (Aim 1). We further propose to assess the functional effects of 5?-tRNA halves/cP- RNAs on cellular focal adhesion, migration, proliferation, and morphology of ASM cells (Aim 2) and to investigate the molecular mechanisms underlying the functional effects of those RNAs (Aim 3). The proposed studies will reveal a novel ncRNA-engaged pathway in asthma pathogenesis and will support the exploration of biomarkers in asthma.